Changes in Fluorescence Recovery After Photobleaching (FRAP) as an indicator of SOX9 transcription factor activity

Govindaraj, Kannan; Hendriks, Jan; Lidke, Diane S.; Karperien, Marcel; Post, Janine N.

doi:10.1016/j.bbagrm.2018.11.001

Cited by 15 publications

(22 citation statements)

References 47 publications

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“…The vector expressing SOX9-mGFP was constructed by cloning mGFP (PS100040, Origene) with the C-terminal of wild type SOX9 (RC208944, Origene) using SgfI and MluI restriction sites. The correct reading frame of the fusion construct was verified by sequencing and its functionality was confirmed by mutation studies [13].…”

Section: Vector-plasmidsmentioning

confidence: 95%

“…We have previously shown that the average TF-FRAP rates correlate to DNA binding (ChIP assay) and transcriptional activity (gene expression analysis) [13]. However, these bulk measurements did not give sufficient information about SOX9 transcriptional activity in a time-resolved manner at the subpopulation level in a heterogenic cell population in, for example, MSCs.…”

Section: Introductionmentioning

confidence: 99%

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Mapping SOX9 transcriptional dynamics during multi-lineage differentiation of human mesenchymal stem cells

Govindaraj

Khurana

Karperien

et al. 2021

Preprint

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The master transcription factor SOX9 is a key player during chondrocyte differentiation, cartilage development, homeostasis and disease. Modulation of SOX9 and its target gene expression is essential during chondrogenic, osteogenic and adipogenic differentiation of human mesenchymal stem cells (hMSCs). However, lack of sufficient knowledge about the signaling interplay during differentiation remains one of the main reasons preventing successful application of hMSCs in regenerative medicine. We previously showed that Transcription Factor – Fluorescence Recovery After Photobleaching (TF-FRAP) can be used to study SOX9 dynamics at the single cell level. We showed that changes in SOX9 dynamics are linked to its transcriptional activity. Here, we investigated SOX9 dynamics during differentiation of hMSCs into the chondrogenic, osteogenic and adipogenic lineages. We show that there are clusters of cells in hMSCs with distinct SOX9 dynamics, indicating that there are a number of subpopulations present in the heterogeneous hMSCs. SOX9 dynamics data at the single cell resolution revealed novel insights about its activity in these subpopulations (cell types). In addition, the response of SOX9 to differentiation stimuli varied in these subpopulations. Moreover, we identified donor specific differences in the number of cells per cluster in undifferentiated hMSCs, and this correlated to their differentiation potential.

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Section: Vector-plasmidsmentioning

confidence: 95%

Section: Introductionmentioning

confidence: 99%

Mapping SOX9 transcriptional dynamics during multi-lineage differentiation of human mesenchymal stem cells

Govindaraj

Khurana

Karperien

et al. 2021

Preprint

Self Cite

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“…Fluorescence recovery after photo bleaching (FRAP) utilizes time-lapse imaging and a fluorescently tagged molecule to determine mobility of proteins within a given system. This approach has been widely used to study protein dynamics in the context of living cells ( van Royen et al., 2009 ) as multiple studies have found a strong link between transcription factor mobility and transcription factor function ( Govindaraj et al., 2019 ; Stenoien et al., 2001a ). Previous FRAP on ERα ( Amita et al., 2010 ; Damdimopoulos et al., 2008 ) demonstrated a rapidly moving receptor within the nucleus under control conditions (no ligand added) and decreased mobility upon E2 or 4HT treatment, owing, in part, to the receptor binding to DNA, protein-protein interactions, and ill-defined nuclear structures (i.e., nucleoskeleton/nuclear matrix).…”

Section: Introductionmentioning

confidence: 99%

Endocrine disrupting chemicals differentially alter intranuclear dynamics and transcriptional activation of estrogen receptor-α

et al. 2021

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Summary Transcription is a highly regulated sequence of stochastic processes utilizing many regulators, including nuclear receptors (NR) that respond to stimuli. Endocrine disrupting chemicals (EDCs) in the environment can compete with natural ligands for nuclear receptors to alter transcription. As nuclear dynamics can be tightly linked to transcription, it is important to determine how EDCs affect NR mobility. We use an EPA-assembled set of 45 estrogen receptor-α (ERα) ligands and EDCs in our engineered PRL-Array model to characterize their effect upon transcription using fluorescence in situ hybridization and fluorescence recovery after photobleaching (FRAP). We identified 36 compounds that target ERα-GFP to a transcriptionally active, visible locus. Using a novel method for multi-region FRAP analysis we find a strong negative correlation between ERα mobility and inverse agonists. Our findings indicate that ERα mobility is not solely tied to transcription but affected highly by the chemical class binding the receptor.

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“…Next, we assessed the distribution of GFP-Ubx populations revealing a larger immobile population of GFP-Ubx WT compared to GFP-Ubx N51A (Fig.7b). The immobile population thus refers to the Ubx WT molecule stably bound to enhancers and promoters (Govindaraj et al, 2019). Notably, the residual immobile population observed for the GFP-Ubx N51A could account for the protein clusters observed for both GFP-Ubx forms (Supplementary Fig.13c).…”

Section: Resultsmentioning

confidence: 99%

The Hox transcription factor Ultrabithorax binds RNA and regulates co-transcriptional splicing through an interplay with RNA polymerase II

Carnesecchi

Boumpas

et al. 2021

Preprint

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Transcription Factors (TFs) play a pivotal role in cell fate decision by coordinating distinct gene expression programs. Although most TFs act at the DNA regulatory layer, few TFs can bind RNA and modulate mRNA splicing. Yet, the mechanistic cues underlying TFs function in splicing remain elusive. Focusing on the Drosophila Hox TF Ultrabithorax (Ubx), our work shed light on a novel layer of Ubx function at the RNA level. Transcriptome and genome-wide binding profiles in embryonic mesoderm and Drosophila cells indicate that Ubx regulates mRNA expression and splicing to promote distinct functions in defined cellular contexts. Ubx modulates splicing via its DNA-binding domain, the Homeodomain (HD). Our results demonstrate a new RNA-binding ability of Ubx in cells and in vitro. Notably, the N51 amino acid of the HD, which mediates Ubx-DNA interaction, is non-essential for Ubx-RNA interaction in vitro but is required in vivo. We find that the N51 amino acid is necessary to mediate interaction between Ubx and the active form of the RNA Polymerase II (Pol II S2Phos) in Drosophila cells. By combining molecular and imaging approaches, our results reveal that Ubx mediates elongation-coupled splicing via a dynamic interplay with active Pol II and chromatin binding. Overall, our work uncovered a novel role of the Hox TFs at the mRNA regulatory layer. This could be an essential function for other classes of TFs to control cell diversity.

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Changes in Fluorescence Recovery After Photobleaching (FRAP) as an indicator of SOX9 transcription factor activity

Cited by 15 publications

References 47 publications

Mapping SOX9 transcriptional dynamics during multi-lineage differentiation of human mesenchymal stem cells

Mapping SOX9 transcriptional dynamics during multi-lineage differentiation of human mesenchymal stem cells

Endocrine disrupting chemicals differentially alter intranuclear dynamics and transcriptional activation of estrogen receptor-α

The Hox transcription factor Ultrabithorax binds RNA and regulates co-transcriptional splicing through an interplay with RNA polymerase II

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